Our long-term goal is to develop a safe and efficacious vaccine for human immune-deficiency virus using the simian immune-deficiency virus (SIV) infection of rhesus macaques as a model. In our parent grant, we hypothesized that vaccinia virus (VV) recombinants expressing SIV antigens and interferon-gamma (IFN-gamma) are safe and effective, eliciting protective immune responses against SIV. Since then, we have advanced our studies on W vaccine development considerably. First, we found that deletion of the VV IFN-gamma receptor homologue (B8R) gene enhances W vector safety without a concomitant reduction in immuno-genicity. Second, we developed a new-generation recombinant VV (rVV) vaccine for rinderpest (v2RVFH) that expresses both glycoprotein genes of rinderpest virus under strong synthetic W promoters. Unlike its predecessors, v2RVFH expresses high levels of the glycoproteins and provides cattle with long-term sterilizing immunity. Finally, we have successfully vaccinated macaques orally with an rVV expressing human IFN-gamma, even though the cytokine greatly attenuates the vector. We now propose to develop a new rW vaccine for SIV, employing our latest generation of vectors, that has a number of advantages compared to the one in the 3arent grant: 1) levels of expression of SIV antigens will be enhanced with the use of strong synthetic 3romoters; 2) the pol gene will be expressed in addition to gag, env, and nef; 3) the macaque IFN-gamma gene will be used in place of the human IFN-gamma gene; and 4) the VV thymidine kinase (a neurovirulence factor) and B8R genes will be insertionally inactivated to further enhance safety. As an added bonus, the B8R protein will not be able to inactivate the expressed IFN-gamma. Our hypothesis is that oral immunization with rWs having enhanced safety and efficacy will elicit a protective immune response against mucosal challenge with pathogenic virus. The goals of this R21 application are to develop the new rW vector and, in conjunction with ongoing experiments supported by the parent grant, assess its safety and efficacy in macaques immunized orally with the rVV and challenged with SIV by mucosal route.